Biomolecular nanotechnology is one of the emerging fields to design biological structures on natural scale. The technology includes using DNA, RNA, proteins, peptides, carbohydrates as templates for providing nanomaterials as multivalent scaffolds for drug delivery, enzyme inhibition and for vaccine development, glycan related biological and medical problems.
Self assembling of the biomaterials particularly composed of nucleic acids, peptides using DNA and RNA, for example, to create nanoshapes and patterns, molecular containers, three dimensional macroscopic crystals are disclosed in U.S. Pat. No. 675,039, US20160122392, U.S. Pat. No. 8,575,110 or U.S. Pat. No. 8,546,337.
Various state-of-the-art nanobiotechnologies for designing supramolecular protein complexes for the development of novel functional nanobiomaterials are discussed in the review article titled ‘Design and construction of self-assembling supramolecular protein complexes using artificial and fusion proteins as nanoscale building blocks’ by Naoya Kobayashi et. al published in Current Opinion in Biotechnology, Volume 46, August 2017, Pages 57-65.
To design proteins to self-assemble into a complex but well defined structure, the protein must contain multiple self assembling interfaces. The protein engineering comprises two general strategies, viz. rational protein design and directed evolution. In the rational protein design the detailed knowledge of the structure and function of the protein is accounted to make desired changes. The technique is relatively inexpensive and technically simple since the method involves site-directed mutagenesis. However, the technique has the major drawback in that in most instances the detailed structural knowledge of protein is often unavailable and further it may sometimes become difficult to predict the effects of various mutations.
In directed evolution, random mutagenesis is applied to a protein, and a selection regime is used to pick out variants that have the desired qualities. The drawback of the method is that it requires high-throughput screening which may not be feasible for all proteins.
Further, the functional group diversity in natural protein is limited to standard 20 amino acids and therefore diversity of protein scaffold is small which limits protein nanotechnology application. Moreover, most of the work related to protein nanotechnology is carried out using standard genetic engineering which is costly.
Hydrophobins, low molecular mass (≤20 kDa) secreted proteins of fungi, are characterized by moderate to high levels of hydrophobicity and the presence of eight conserved cysteine (Cys) residues. The amphiphilic structure possesses both hydrophilic and hydrophobic domains and can self-assemble from a soluble form into an insoluble and amphipathic monolayer at hydrophilic:hydrophobic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic materials. The self-assembling properties and amphipathic nature of hydrophobins make them attractive candidates for biotechnological and medical applications.
Based on differences in hydropathy patterns and biophysical properties, the hydrophobins are classified into two categories viz. class I and class II. The Class I monolayer contains the similar core structure as amyloid fibrils, and is positive to Congo red and thioflavin T. The monolayer formed by class I hydrophobins has a highly ordered structure, and can only be dissociated by concentrated trifluoroacetate or formic acid. Monolayer assembly involves large structural rearrangements with respect to the monomer. The monolayers formed by class II hydrophobins lack the fibrillar rodlet morphology and can be solubilized with organic solvents and detergents.
Hydrophobins can be produced by fermentation of microorganisms (bacteria or fungi) or by fermentation of genetically modified microorganisms. These naturally occurring facially amphiphilic proteins present in micro-organisms and their structural aspects and mechanisms by which they assemble and the advancements in the use of hydrophobins for cell attachment, drug delivery, and protein purification are discussed in the documents; Two Forms and Two Faces, Multiple States and Multiple Uses: Properties and Applications of the Self-Assembling Fungal Hydrophobins by Qin Ren et. al. published on 31 Jul. 2013; Oligomerization of hydrophobin SC3 in solution: From soluble state to self-assembly by Xiaoqin Wang et. al published in Protein Science (2004); Structure-Function Relationships in Hydrophobins: Probing the Role of Charged Side Chains by Michael Lienemann et. al published in Applied and Environmental Microbiology, vol 79, no. 18, p. 5533-5538; Spontaneous self-assembly of SC3 hydrophobins into nanorods in aqueous solution by AgataZykwinska et. al published in Biochimica et BiophysicaActa 1844 (2014) 1231-1237.
During the past decade efforts are made for creating hydrophobins with improved properties and functions by bridging components derived from both natural and synthetic domains, specifically, to synthetic biomimetic packaging of functional proteins. Developing range of well-defined protein-polymer amphiphiles is very critical in clinical medical use such as in drug delivery of hydrophobic drugs, in vaccine development and for encapsulating drugs or bio macromolecules.
WO2012058343 describes protein based conjugates which comprises a globular protein conjugated with a polymer that preserves the folded and functional structure of a protein. The globular protein is covalently or non-covalently attached to the polymer and is selected from the group consisting of mCherry, a green fluorescent protein, lysozyme, albumin, and carbonic anhydrase. The polymer is a synthetic polymer. The conjugates self-assemble into a three dimensional solid-state or gel-state nanostructure.
US2016120814 describes a method for preparing a protein cage comprising; (i) 1st step of preparing an amphiphilic polymer comprising a 1st hydrophobic polymer and a 1st hydrophilic functional group; (ii) a 2nd step of preparing a hydrophilic protein comprising a 2nd functional group binding to the 1st functional group; (iii) a 3rd step of forming an amphiphilic polymer-protein hybrid by the binding of the 1st functional group and the 2nd functional group, and forming core-shell structured particles comprising a protein shell and an amphiphilic polymer core by the self-assembly of the amphiphilic polymer in a hydrophilic solvent; and (iv) a 4th step of removing some or all of the hydrophobic polymer of the core part from the core-shell structured particles. Example 1 illustrates the synthetic mechanism of the polymer bound to the Ni-NTA (Nitrilotriacetic acid) terminal as shown in the scheme below:

Article titled “Chemical Strategies for the Synthesis of Protein—Polymer Conjugates” by Bjorn Jung and Patrick Theato published in AdvPolymSci (2013) 253: 37-70; DOI: 10.1007/12_2012_169 reviews the numerous chemical strategies that have been developed to conjugate different synthetic polymers onto protein surfaces (deriving from selected amino acid residues), which are advantageous in biomedical applications.
In spite of the developments in the field of polymer directed protein assemblies there remains a need in the art to provide novel semi synthetic hydrophobin mimics that can self-assemble to protein nano container in a specified chemical environment for use in bio-nanotechnology.
The other objective is to expedite synthesis of hydrophobin mimics of different sizes and shapes with high precision.
Yet another objective is to create a library of hydrophobin mimics by tuning the protein head group, linker and hydrophobic part synthetically which is difficult to achieve in other reported method to cater to the needs of bio-nanotechnology.